Refolding Proteins Using a Chemically Controlled Redox State

ABSTRACT

A method of refolding proteins expressed in non-mammalian cells present in concentrations of 2.0 g/L, or higher is disclosed. The method comprises identifying the thiol pair ratio and the redox butler strength to achieve conditions under which efficient folding at concentrations of 2.0 g/L or higher is achieved and can be employed over a range of volumes, including commercial scale.

This application claims the benefit of U.S. Provisional Application No.61/219,257 filed Jun. 22, 2009, which is incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention generally relates to refolding proteins at highconcentrations, and more particularly to refolding proteins in volumesat concentrations of 2.0 g/L and above.

BACKGROUND OF THE INVENTION

Recombinant proteins can be expressed in a variety of expressionsystems, including non-mammalian cells, such as bacteria and yeast. Adifficulty associated with the expression of recombinant proteins inprokaryotic cells, such as bacteria, is the precipitation of theexpressed proteins in limited-solubility intracellular precipitatestypically referred to as inclusion bodies. Inclusion bodies are formedas a result of the inability of a bacterial host cell to foldrecombinant proteins properly at high levels of expression and as aconsequence the proteins become insoluble. This is particularly true ofprokaryotic expression of large, complex or protein sequences ofeukaryotic origin. Formation of incorrectly folded recombinant proteinshas, to an extent, limited the commercial utility of bacterialfermentation to produce recombinant large, complex proteins, at highlevels of efficiency.

Since the advent of the recombinant expression of proteins atcommercially viable levels in non-mammalian expression systems such asbacteria, various methods have been developed for obtaining correctlyfolded proteins from bacterial inclusion bodies. These methods generallyfollow the procedure of expressing the protein, which typicallyprecipitates in inclusion bodies, lysing the cells, collecting theinclusion bodies and then solubilizing the inclusion bodies in asolubilization buffer comprising a denaturant or surfactant andoptionally a reductant, which unfolds the proteins and disassembles theinclusion bodies into individual protein chains with little to nostructure. Subsequently, the protein chains are diluted into or washedwith a refolding buffer that supports renaturation to a biologicallyactive form. When cysteine residues are present in the primary aminoacid sequence of the protein, it is often necessary to accomplish therefolding in an environment which allows correct formation of disulfidebonds (e.g., a redox system).

Typical refold concentrations for complex molecules, such as moleculescomprising two or more disulfides, are less than 2.0 g/L and moretypically 0.01-0.5 g/L (Rudolph & Lilie, (1996) FASEB J. 10:49-56).Thus, refolding large masses of a complex protein, such as an antibody,peptibody or other Fc fusion protein, at industrial production scalesposes significant limitations due to the large volumes required torefold proteins, at these typical product concentration, and is a commonproblem facing the industry. One factor that limits the refoldconcentration of these types of proteins is the formation of incorrectlypaired disulfide bonds, which may in turn increase the propensity forthose forms of the protein to aggregate. Due to the large volumes ofmaterial and large pool sizes involved when working with industrialscale protein production, significant time, and resources can be savedby eliminating or simplifying one or more steps in the process.

While protein refolding has previously been demonstrated at higherconcentrations, the proteins that were refolded were eithersignificantly smaller in molecular weight, less complex moleculescontaining only one or two disulfide bonds (see, e.g., Creighton, (1974)J. Mol. Biol. 87:563-577). Additionally, the refolding processes forsuch proteins employed detergent-based refolding chemistries (see, e.g.,Stöckel et al., (1997) Eur J Biochem 248:684-691) or utilized highpressure folding strategies (St John et al., (2001) J. Biol. Chem.276(50):46856-63). More complex molecules, such as antibodies,peptibodies and other large proteins, are generally not amenable todetergent refold conditions and are typically refolded in chaotropicrefold solutions. These more complex molecules often have greater thantwo disulfide bonds, often between 8 and 24 disulfide bonds, and can bemulti-chain proteins that form homo- or hetero-dimers.

Until the present disclosure, these types of complex molecules could notbe refolded at high concentrations, i.e., concentrations of 2.0 g/L andhigher, with any meaningful degree of efficiency on a small scale, andnotably not on an industrial scale. The disclosed methods, in contrast,can be performed at high concentrations on a small or large (e.g,industrial) scale to provide properly refolded complex proteins. Theability to refold proteins at high concentrations and at large scalescan translate into not only enhanced efficiency of the refold operationitself, but also represents time and cost savings by eliminating theneed for additional equipment and personnel. Accordingly, a method ofrefolding proteins present in high concentrations could translate intohigher efficiencies and cost savings to a protein production process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of plots depicting depicting the effect of thiol-pairratio and redox buffer strength on product-species distribution; FIG. 1a depicts the effect of a 5 mM buffer strength; FIG 1 b depicts theeffect of a 7.5 mM buffer strength; FIG. 1 c depicts the effect of a 10mM buffer strength; FIG. 1 d depicts the effect of a 12.5 mM bufferstrength; FIG. 1 e depicts the effect of a 15 mM buffer strength andFIG. 1 f depicts the effect of a 20 mM buffer strength.

FIG. 2 is a series of plots depicting the effect of the degree ofaeration on the species distribution under fixed thiol-pair ratio andthiol-pair buffer strength.

FIG. 3 is an analytical overlay of a chemically controlled, non-aerobicrefold performed at 6 g/L and optimized using an embodiment of thedescribed method performed at 1 L and 2000 L.

SUMMARY OF THE INVENTION

A method of refolding a protein expressed in a non-mammalian expressionsystem and present in a volume at a concentration of 2.0 g/L or greatercomprising: (a) contacting the protein with a refold buffer comprising aredox component comprising a final thiol-pair ratio having a range of0.001 to 100 and a redox buffer strength of 2 mM or greater and one ormore of: (i) a denaturant; (ii) an aggregation suppressor; and (iii) aprotein stabilizer; to form a refold mixture; (b) incubating the refoldmixture; and (c) isolating the protein from the refold mixture.

In various embodiments the redox component has a final thiol-pair ratiogreater than or equal to 0.001 but less than or equal to 100, forexample within a range of 0.05 to 50, 0.1 to 50, 0.25 to 50, 0.5 to 50,0.75 to 40, 1.0 to 50 or 1.5 to 50, 2 to 50, 5 to 50, 10 to 50, 15 to50, 20 to 50, 30 to 50 or 40 to 50 and a Thiol-pair buffer strengthequal to or greater than 2 mM, for example greater than or equal to 2.25mM, 2.5 mM, 2.75 mM, 3 mM, 5 mM, 7.5 mM, 10 mM, or 15 mM, wherein thethiol-pair buffer strength is effectively bounded at a maximum of 100mM. Restated, in terms of ranges, the thiol buffer strength can bebetween 2 and 20 mM, for example between 2.25 mM and 20 mM, 2.5 mM and20 mM, 2.75 mM and 20 mM, 3 mM and 20 mM, 5 mM and 20 mM, 7.5 mM and 20mM, 10 mM and 20 mM, or 15 mM and 20 mM, to form a mixture.

In one embodiment of a refold buffer, the refold buffer comprises urea,arginine-HCl, cysteine and cystamine in Tris buffer. In a furtherembodiment the components are present in the refold buffer inproportions described in Example 3.

In another embodiment of a refold buffer, the refold buffer comprisesurea, arginine HCl, glycerol, cysteine, and cystamine in Iris buffer. Ina further embodiment the components are present in the refold buffer inproportions described in Example 4.

In some embodiments, the protein is initially present in a volume in anon-native limited solubility form, such as an inclusion body.Alternatively, the protein is present in the volume in a soluble form.The protein can be a recombinant protein or it can be an endogenousprotein. The protein can be a complex protein such as an antibody or amultimeric protein. In another embodiment, the protein is an Fc-proteinconjugate, such as a protein fused or linked to a Fc domain.

The non-mammalian expression system can be a bacterial expression systemor a yeast expression system.

The denaturant in the refold buffer can be selected from the groupconsisting of urea, guanidinium salts, dimethyl urea, methylurea andethylurea. The protein stabilizer in the refold buffer can be selectedfrom the group consisting of arginine, proline, polyethylene glycols,non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol,sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate andosmolytes. The aggregation suppressor can be selected from the groupconsisting of arginine, proline, polyethylene glycols, non-ionicsurfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose,sorbitol, glucose, Tris, sodium sulfate, potassium sulfate andosmolytes. The thiol-pairs can comprise at least one component selectedfrom the group consisting of glutathione-reduced, glutathione-oxidized,cysteine, cystine, cysteamine, cystamine and beta-mercaptoethanol.

In various embodiments, the purification can comprise contacting themixture with an affinity separation matrix, such as a Protein A orProtein G resin. Alternatively, the affinity resin can be a mixed modeseparation matrix or an ion exchange separation matrix. In variousaspects, the incubation can be performed under aerobic conditions orunder non-aerobic conditions.

DETAILED DESCRIPTION OF THE INVENTION

The relevant literature suggests that when optimizing various proteinrefolding operations, the refold buffer thiol-pair ratio has beenpurposefully varied and as a result the thiol buffer strength wasunknowingly varied across a wide range of strengths (see, e.g., Lilie,Schwarz & Rudolph, (1998) Current Opinion in Biotechnology 9(5):497-501,and Tran-Moseman, Schauer & Clark (1999) Protein Expression &Purification 16(1):181-189). In one study, a relationship between thethiol pair ratio and the buffer strength was investigated for lysozyme,a simple, single-chain protein that forms a molten globule. (DeBernardez et al., (1998) Biotechnol. Prog. 14:47-54). The De Bernardezwork described thiol concentration in terms of a model that consideredonly the kinetics of a one-way reaction model. However, most complexproteins are governed by reversible thermodynamic equilibria that arenot as easily described (see, e.g., Darby et al., (1995) J. Mol. Biol.249:463-477). More complex behavior is expected in the case of largemulti-chain proteins containing many disulfide bonds, such asantibodies, peptibodies and other Fc fusion proteins. Until the presentdisclosure, specific relationships had not been provided for thiolbuffer strength, thiol-pair ratio chemistry, and protein concentrationwith respect to complex proteins that related to the efficiency ofprotein production. Consequently, the ability to refold proteins in ahighly concentrated volume has largely been an inefficient orunachievable goal, leading to bottlenecks in protein production,particularly on the industrial scale.

Prior to the present disclosure a specific controlled investigation ofthe independent effects of thiol-pair ratio and thiol-pair bufferstrength had not been disclosed for complex proteins. As describedherein, by controlling the thiol-pair buffer strength, in conjunctionwith thiol-pair ratio and protein concentration, the efficiency ofprotein folding operations can be optimized and enhanced and therefolding of proteins at high concentrations, for example 2 g/L orgreater, can be achieved.

Thus, in one aspect, the present disclosure relates to theidentification and control of redox thiol-pair ratio chemistries thatfacilitate protein refolding at high protein concentrations, such asconcentrations higher than 2.0 g/L. The method can be applied to anytype of protein, including simple proteins and complex proteins (e.g.,proteins comprising 2-23 disulfide bonds or greater than 250 amino acidresidues, or having a MW of greater than 20,000 daltons), includingproteins comprising a Fc domain, such as antibodies, peptibodies andother Fc fusion proteins, and can be performed on a laboratory scale(typically milliliter or liter scale), a pilot plant scale (typicallyhundreds of liters) or an industrial scale (typically thousands ofliters). Examples of complex molecules known as peptibodies, and otherFc fusions, are described in U.S. Pat. No. 6,660,843, U.S. Pat. No.7,138,370 and U.S. Pat. No. 7,511,012.

As described herein, the relationship between thiol buffer strength andredox thiol-pair ratio has been investigated and optimized in order toprovide a reproducible method of refolding proteins at concentrations of2.0 g/L and higher on a variety of scales. A mathematical formula wasdeduced to allow the precise calculation of the ratios and strengths ofindividual redox couple components to achieve matrices of bufferthiol-pair ratio and buffer thiol strength. Once this relationship wasestablished, it was possible to systematically demonstrate that thiolbuffer strength and the thiol-pair ratio interact to define thedistribution of resulting product-related species in a refoldingreaction.

The buffer thiol-pair ratio is, however, only one component indetermining the total system thiol-pair ratio in the total reaction.Since the cysteine residues in the unfolded protein are reactants aswell, the buffer thiol strength needs to vary in proportion withincreases in protein concentration to achieve the optimal systemthiol-pair ratio. Thus, in addition to demonstrating that buffer thiolstrength interacts with the thiol-pair ratio, it has also been shownthat the buffer thiol strength relates to the protein concentration inthe total reaction as well. Optimization of the buffer thiol strengthand the system thiol pair ratio can be tailored to a particular protein,such as a complex protein, to minimize cysteine mispairing yet stillfacilitate a refold at a high concentration.

I. Definitions

As used herein, the terms “a” and “an” mean one or more unlessspecifically indicated otherwise.

As used herein, the term “non-mammalian expression system” means asystem for expressing proteins in cells derived from an organism otherthan a mammal, including but not limited to, prokaryotes, includingbacteria such as E. coli, and yeast. Often a non-mammalian expressionsystem is employed to express a recombinant protein of interest, whilein other instances a protein of interest is an endogenous protein thatis expressed by a non-mammalian cell. For purposes of the presentdisclosure, regardless of whether a protein of interest is endogenous orrecombinant, if the protein is expressed in a non-mammalian cell thenthat cell is a “non-mammalian expression system.” Similarly, a“non-mammalian cell” is a cell derived from an organism other than amammal, examples of which include bacteria or yeast.

As used herein, the term “denaturant” means any compound having theability to remove some or all of a protein's secondary and tertiarystructure when placed in contact with the protein. The term denaturantrefers to particular chemical compounds that affect denaturation, aswell as solutions comprising a particular compound that affectdenaturation. Examples of denaturants that can be employed in thedisclosed method include, but are not limited to urea, guanidiniumsalts, dimethyl urea, methylurea, ethylurea and combinations thereof.

As used herein, the term “aggregation suppressor” means any compoundhaving the ability to disrupt and decrease or eliminate interactionsbetween two or more proteins. Examples of aggregation suppressors caninclude, but are not limited to, amino acids such as arginine, proline,and glycine; polyols and sugars such as glycerol, sorbitol, sucrose, andtrehalose; surfactants such as, polysorbate-20, CHAPS, Triton X-100, anddodecyl maitoside; and combinations thereof.

As used herein, the term “protein stabilizer” means any compound havingthe ability to change a protein's reaction equilibrium state, such thatthe native state of the protein is improved or favored. Examples ofprotein stabilizers can include, but are not limited to, sugars andpolyhedric alcohols such as glycerol or sorbitol; polymers such aspolyethylene glycol (PEG) and α-cyclodextrin; amino acids salts such asarginine, proline, and glycine; osmolytes and certain Hoffmeister saltssuch as Tris, sodium sulfate and potassium sulfate; and combinationsthereof.

As used herein, the terms “Fc” and “Fc region” are used interchangeablyand mean a fragment of an antibody that comprises human or non-human(e.g., murine) C_(H2) and C_(H3) immunoglobulin domains, or whichcomprises two contiguous regions which are at least 90% identical tohuman or non-human C_(H2) and C_(H3) immunoglobulin domains. An Fc canbut need not have the ability to interact with an Fc receptor. See,e.g., Hasemann & Capra, “Immunoglobulins: Structure and Function,” inWilliam E. Paul, ed., Fundamental immunology, Second Edition, 209,210-218 (1989), which is incorporated by reference herein in itsentirety.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and mean any chain of at least five naturally ornon-naturally occurring amino acids linked by peptide bonds.

As used herein, the terms “isolated” and “purify” are usedinterchangeably and mean to reduce by 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or95%, or more, the amount of heterogenous elements, for examplebiological macromolecules such as proteins or DNA, that may be presentin a sample comprising a protein of interest. The presence ofheterogenous proteins can be assayed by any appropriate method includingHigh-performance Liquid Chromatography (HPLC), gel electrophoresis andstaining and/or ELISA assay. The presence of DNA and other nucleic acidscan be assayed by any appropriate method including gel electrophoresisand staining and/or assays employing polymerase chain reaction.

As used herein, the term “complex molecule” means any protein that is(a) larger than 20,000 MW, or comprises greater than 250 amino acidresidues, and (b) comprises two or more disulfide bonds in its nativeform. A complex molecule can, but need not, form multimers. Examples ofcomplex molecules include but are not limited to, antibodies,peptibodies and other chimeric molecules comprising an Fc domain andother large proteins. Examples of complex molecules known aspeptibodies, and other Fc fusions, are described in U.S. Pat. No.6,660,843, U.S. Pat. No. 7,138,370 and U.S. Pat. No. 7,511,012.

As used herein, the term “peptibody” refers to a polypeptide comprisingone or more bioactive peptides joined together, optionally via linkers,with an Fe domain. See U.S. Pat. No. 6,660,843, U.S. Pat. No. 7,138,370and U.S. Pat. No. 7,511,012 for examples of peptibodies.

As used herein, the term “refolding” means a process of reintroducingsecondary and tertiary structure to a protein that has had some or allof its native secondary or tertiary structure removed, either in vitroor in vivo, e.g., as a result of expression conditions or intentionaldenaturation and/or reduction. Thus, a refolded protein is a proteinthat has had some or all of its native secondary or tertiary structurereintroduced.

As used herein, the term “buffer thiol-pair ratio” is defined by therelationship of the reduced and oxidized redox species used in theretold butter as defined in Equation 1:

Equation 1 Definition of Buffer Thiol-Pair Ratio (TPR)

${{Buffer}\mspace{14mu} T\; P\; R} = {\frac{\lbrack{reductant}\rbrack^{2}}{\lbrack{oxidant}\rbrack} = \frac{\lbrack{cysteine}\rbrack^{2}}{\lbrack{cystamine}\rbrack}}$

As used herein, the terms “Buffer Thiol Strength”, “Thiol-Pair BufferStrength”, and “Thiol-pair Strength” are used interchangeably and aredefined in Equation 2, namely' as the total mono-equivalent thiolconcentration, wherein the total concentration is the sum of the reducedspecies and twice the concentration of the oxidized species.

Equation 2 Definition of Buffer Thiol-Pair Buffer Strength/Thiol BufferStrength (BS)

Thiol-Pair Buffer Strength=2 oxidand+keductand=2[cystamine]+[cysteine]

The relationship between the thiol-pair ratio and thiol-pair bufferstrength is described in equations 3 and 4.

Equation 3 Calculation of the Reduced Redox Species with Regard to aDefined Redox Buffer Strength (BS) and buffer Redox Potential

${{Concentrat}{\; \;}{i{on}}\mspace{14mu} {of}\mspace{14mu} {Reduced}\mspace{14mu} {Redox}\mspace{14mu} {Component}} = \frac{( \sqrt{{{bufferT}\; P\; R^{2}} + {8*{bufferT}\; P\; R*B\; S}} ) - {{bufferT}\; P\; R}}{4}$

Equation 4 Calculation of the Oxidized Redox Species with Regard to aDefined Redox Buffer Strength (BS) and Buffer Redox Potential

${{Concentration}\mspace{14mu} {of}\mspace{14mu} {Oxidized}\mspace{14mu} {Redox}\mspace{14mu} {Component}} = \frac{( {{Concentration}\mspace{14mu} {of}\mspace{14mu} {Reduced}\mspace{14mu} {Redox}\mspace{14mu} {Component}} )^{2}}{T\; P\; R}$

As used herein, the term “redox component” means any thiol-reactivechemical or solution comprising such a chemical that facilitates areversible thiol exchange with another thiol or the cysteine residues ofa protein. Examples of such compounds include, but are not limited to,glutathione-reduced, glutathione-oxidized, cysteine, cystine,cysteamine, cystamine, beta-mercaptoethanol and combinations thereof.

As used herein, the term “solubilization” means a process in whichsalts, ions, denaturants, detergents, reductants and/or other organicmolecules are added to a solution comprising a protein of interest,thereby removing some or all of a protein's secondary and/or tertiarystructure and dissolving the protein into the solvent. This process caninclude the use of elevated temperatures, typically 10-50° C., but moretypically 15-25° C., and/or alkaline pH, such as pH 7-12. Solubilizationcan also be accomplished by the addition of acids, such as 70% formicacid (see, e.g., Cowley & Mackin (1997) FEBS Lett 402:124-130).

A “solubilized protein” is a protein in which sonic or all of theprotein's secondary and/or tertiary structure has been removed.

A “solublization pool” is a volume of solution comprising a solubilizedprotein of interest as well as the salts, ions, denaturants, detergents,reductants and/or other organic molecules selected to solubilize theprotein.

As used herein, the term “non-aerobic condition” means any reaction orincubation condition that is performed without the intentional aerationof the mixture by mechanical or chemical means. Under non-aerobicconditions oxygen can be present, as long as it is naturally present andwas not introduced into the system with the intention of adding oxygento the system. Non-aerobic conditions can be achieved by, for example,limiting oxygen transfer to a reaction solution by limiting headspacepressure, the absence of, or limited exposure to, air or Oxygencontained in the holding vessel, air or oxygen overlay, the lack ofspecial accommodations to account for mass transfer during processscaling, or the absence of gas sparging or mixing to encourage thepresence of oxygen in the reaction system. Non-aerobic conditions canalso be achieved by intentionally limiting or removing oxygen from thesystem via chemical treatment, headspace overlays or pressurization withinert gases or vacuums, or by sparging with gases such as argon ornitrogen, results in the reduction of oxygen concentration in thereaction mixture.

As used herein, the terms “non-native” and “non-native form” are usedinterchangeably and when used in the context of a protein of interest,such as a protein comprising a Fc domain, mean that the protein lacks atleast one formed structure attribute found in a form of the protein thatis biologically active in an appropriate in vivo or in vitro assaydesigned to assess the protein's biological activity. Examples ofstructural features that can be lacking in a non-native form of aprotein can include, but are not limited to, a disulfide bond,quaternary structure, disrupted secondary or tertiary structure or astate that makes the protein biologically inactive in an appropriateassay. A protein in a non-native form can but need not form aggregates.

As used herein, the term “non-native limited solubility form” when usedin the context of a protein of interest, such as a protein comprising aFc domain, means any form or state in which the protein lacks at leastone formed structural feature found in a form of the protein that (a) isbiologically active in an appropriate in vivo or in vitro assay designedto assess the protein's biological activity and/or (b) forms aggregatesthat require treatment, such as chemical treatment, to become soluble.The term specifically includes proteins existing in inclusion bodies,such as those sometimes found when a recombinant protein is expressed ina non-mammalian expression system.

II. Theory

Refolding microbial-derived molecules present in a pool atconcentrations of 2.0 g/L or higher is advantageous for a variety ofreasons, primarily because of the associated reduction in reactionvolumes and increases in process throughput. From a process scalingstandpoint, it is advantageous to refold under conditions that do notrequire aerobic conditions; such conditions can be achieved, forexample, by constant or intermittent sparging, the implementation of airor oxygen headspace overlays, by pressurizing the headspace, or byemploying high efficiency mixing. Since the oxygen concentration in thesystem is related to mass transfer, the scaling of the refold reactionbecomes considerably more difficult as factors such as tank geometry,volume, and mixing change. Furthermore, oxygen may not be a directreactant in the formation of disulfide bonds in the protein, making adirect link to the mass transfer coefficient unlikely. This furthercomplicates scaling of the reaction. Therefore, non-aerobic, chemicallycontrolled redox systems are preferred for refolding proteins. Examplesof such conditions are provided herein.

The optimal refold chemistry for a given protein represents a carefulbalance that maximizes the folded/oxidized state while minimizingundesirable product species, such as aggregates, unformed disulfidebridges (e.g., reduced cysteine pairs), incorrect disulfide pairings(which can lead to misfolds), oxidized amino acid residues, deamidatedamino acid residues, incorrect secondary structure, and product-relatedadducts (e.g., cysteine or cysteamine adducts). One factor that isimportant in achieving this balance is the redox-state of the refoldsystem. The redox-state is affected by many factors, including, but notlimited to, the number of cysteine residues contained in the protein,the ratio and concentration of the redox couple chemicals in the refoldsolution (e.g., cysteine, cystine, cystamine, cysteamine,glutathione-reduced and glutathione-oxidized), the concentration ofreductant carried over from the solubilization buffer (e.g., DTT,glutathione and beta-mercaptoethanol), the level of heavy metals in themixture, and the concentration of oxygen in the solution.

Thiol-pair ratio and thiol-pair buffer strength are defined in Equations1 and 2, infra, using cysteine and cystamine as an example reductant andoxidant, respectively. These quantities, coupled with proteinconcentration and reductant carry-over from the solubilization, can befactors in achieving a balance between the thiol-pair ratio and thethiol-pair buffer strength.

Turning to FIG. 1, this figure depicts the effect of thiol-pair ratioand thiol buffer strength on the distribution of product-relatedspecies, as visualized by reversed phase-HPLC analysis, for a complexchimeric protein. In FIGS. 1 a-1 f, the dotted lines represent proteinspecies with oxidized amino acid residues, single chain species, andstable mixed disulfide intermediates, the dashed lines representmis-paired or incorrectly formed disulfide protein species and proteinspecies with partially unformed disulfide linkages. The solid linesrepresent properly folded protein species. FIGS. 1 a-1 f demonstratethat at a constant 6 g/L protein concentration, as the thiol-pair bufferstrength is increased, the thiol-pair ratio required to achieve acomparable species distribution must also increase. For example, asshown in FIG. 1, if the buffer strength is increased to 10 mM, from 5mM, the balanced thiol-pair ratio would be about 2-fold higher, toachieve a comparable species distribution. This is largely due toincreased buffering of the reductant carried over from thesolubilization, on the total system thiol-pair ratio. At lower redoxbuffer strengths, the overall system becomes much more difficult tocontrol. The protein concentration and number of cysteines contained inthe protein sequence also relate to the minimum required thiol-pairbuffer strength required to control the system. Below a certain point,which will vary from protein to protein, the protein thiol concentrationcan overwhelm the redox couple chemistry and lead to irreproducibleresults.

In the results depicted in FIG. 1, when the thiol-pair ratio of therefolding solution is intentionally set to be more reducing, theresultant product distribution shifts to produce more of the reducedproduct species (dashed lines). When the Thiol-Pair Ratio of therefolding solution is intentionally set to be lower, or more oxidizing,the resultant product distribution shifts to produce more oxidizedresidues, single chain forms, and stable mixed disulfide intermediatespecies (dotted lines). The ability to select an optimal Thiol-PairRatio and Thiol-pair Buffer Strength allows for the optimization of theyield of a desired folded protein form. This optimized yield can beachieved by maximizing the mass or yield of desired folded proteinspecies in the refolding pool or by purposefully shifting the resultantundesired product-related species to a form that is most readily removedin the subsequent purification steps and thusly leads to an overallbenefit to process yield or purity.

Optimization of the redox component Thiol-pair Ratios and Thiol-pairBuffer Strengths can be performed for each protein. A matrix or seriesof multifactorial matrices can be evaluated to optimize the refoldingreaction for conditions that optimize yield and distributions of desiredspecies. An optimization screen can be set up to systematically evaluateredox chemistries, Thiol-pair ratios, Thiol-pair Buffer Strengths,incubation times, protein concentration and pH in a full or partialfactorial matrix, with each component varied over a range of at leastthree concentration or pH levels with all other parameters keptconstant. The completed reactions can be evaluated by RP-HPLC andSE-HPLC analysis for yield and product quality using standardmultivariate statistical tools.

III. Method of Refolding a Protein Expressed in a Non-MammalianExpression System and Present in a Volume at a Concentration of 2.0 g/Lor Greater

The disclosed refold method is particularly useful for refoldingproteins expressed in non-mammalian expression systems. As noted herein,non-mammalian cells can be engineered to produce recombinant proteinsthat are expressed intracellularly in either a soluble or a completelyinsoluble or non-native limited solubility form. Often the cells willdeposit the recombinant proteins into large insoluble or limitedsolubility aggregates called inclusion bodies. However, certain cellgrowth conditions (e.g., temperature or pH) can be modified to drive thecells to produce a recombinant protein in the form of intracellular,soluble monomers. As an alternative to producing proteins in insolubleinclusion bodies, proteins can be expressed as soluble proteins,including proteins comprising an Fc region, which can be captureddirectly from cell lysate by affinity chromatography. Capturing directlyfrom lysate allows for the refolding of relatively pure protein andavoids the very intensive harvesting and separation process that isrequired in inclusion body processes. The refolding method, however, isnot limited to samples that have been affinity purified and can beapplied to any sample comprising a protein that was expressed in anon-mammalian expression system, such as a protein found in a volume ofcell lysate (i.e., a protein that has not been purified in any way).

In one aspect, the present disclosure relates to a method of refolding aprotein expressed in a non-mammalian expression system in a soluble formand present in a volume at a concentration of 2.0 g/L or greater, suchas a protein that has been purified by affinity chromatography from thecell lysate of non-mammalian cells in which the protein was expressed.Although the volume can be derived from any stage of a proteinpurification process, in one example the volume is an affinitychromatography elution pool (e.g., a Protein A elution pool). In anotherexample, the volume is situated in a process stream. The method is notconfined to Fc-containing proteins, however, and can be applied to anykind of peptide or protein that is expressed in a soluble form andcaptured from non-mammalian-derived cell lysate. The isolated solubleprotein is often released from non-mammalian cells in a reduced form andtherefore can be prepared for refolding by addition of a denaturant,such as a chaotrope. Further combination with protein stabilizers,aggregation suppressors and redox components, at an optimized Thiol-pairration and Thiol-pair Buffer Strength, allows for refolding atconcentrations of 1-40 g/L, for example at concentrations of 10-20 g/L.

In one particular embodiment of the method, a protein is expressed in anon-mammalian expression system, and is released from the expressingcell by high pressure lysis. The protein is then captured from thelysate by Protein A affinity chromatography and is present in a volumeat a concentration of 10 g/L or greater. The protein is then contactedwith a refold buffer comprising a denaturant, an aggregation suppressor,a protein stabilizer and a redox component, wherein the redox componenthas a final thiol-pair ratio (as defined herein) having a range of 0.001to 100, for example within a range of 0.05 to 50, 0.1 to 50, 0.25 to 50,0.5 to 50, 0.75 to 40, 1.0 to 50 or 1.5 to 50, 2 to 50, 5 to 50, 10 to50, 15 to 50, 20 to 50, 30 to 50 or 40 to 50 and a Thiol-pair bufferstrength (as defined herein) equal to or greater than 2 mM, for examplegreater than or equal to 2.25 mM, 2.5, 2.75 mM, 3 mM, 5 mM, 7.5 mM, 10mM, or 15 mM, wherein the thiol-pair buffer strength is effectivelybounded at a maximum of 100 mM. Restated, in terms of ranges, the thiolbuffer strength is between 2 and 20 mM, for example between 2.25 mM and20 mM, 2.5 mM and 20 mM, 2.75 mM and 20 mM, 3 mM and 20 mM, 5 mM and 20mM, 7.5 mM and 20 mM, 10 mM and 20 mM, or 15 mM and 20 mM.

In another aspect, the present disclosure relates to a method ofrefolding a protein expressed in a non-mammalian expression system in aninsoluble or limited-solubility form, such as in the form of inclusionbodies. When the protein is disposed in inclusion bodies, the inclusionbodies can be harvested from lysed cells, washed, concentrated andrefolded.

Optimization of the refold buffer can be performed for each protein andeach final protein concentration level using the novel method providedherein. As shown in the Examples, good results can be obtained whenrefolding a protein comprising an Fc region when the refold buffercontains a denaturant (e.g., urea or other chaotrope, organic solvent orstrong detergent), aggregation suppressors (e.g., a mild detergent,arginine or tow concentrations of PEG), protein stabilizers glycerol,sucrose or other osmolyte, salts) and redox components (e.g., cysteine,cystamine, glutathione). The optimal thiol-pair ratio and redox bufferstrength can be determined using an experimental matrix of thiol-pairratio (which can have a range of 0.001 to 100, for example within arange of 0.05 to 50, 0.1 to 50, 0.25 to 50, 0.5 to 50, 0.75 to 40, 1.0to 50 or 1.5 to 50, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 30to 50 or 40 to 50) versus thiol-pair buffer strength (which can begreater than 2 mM, for example greater than or equal to 2.25 mM, 2.5,2.75 mM, 3 mM, 5 mM, 7.5 mM, 10 mM, or 15 mM, wherein the thiol-pairbuffer strength is effectively bounded at a maximum of 100 mM. Restated,in terms of ranges, the thiol buffer strength is between 2 and 20 mM,for example between 2.25 mM and 20 mM, 2.5 mM and 20 mM, 2.75 mM and 20mM, 3 mM and 20 mM, 5 mM and 20 mM, 7.5 mM and 20 mM, 10 mM and 20 mM,or 15 mM and 20 mM, depending on the protein concentration and theconcentration of reductant used to solubilize the inclusion bodies).Conditions can be optimized using the novel methods described in Example2.

In one particular embodiment of the method, a protein is expressed in anon-mammalian expression system and is present in a volume at aconcentration of 2.0 g/L or greater. The protein is contacted with arefold buffer comprising a denaturant, an aggregation suppressor, aprotein stabilizer and a redox component, wherein the redox componenthas a final thiol-pair ratio (as defined herein) having a range of 0.001to 100, for example within a range of 0.05 to 50, 0.1 to 50, 0.25 to 50,0.5 to 50, 0.75 to 40, 1.0 to 50 or 1.5 to 50, 2 to 50, 5 to 50, 10 to50, 15 to 50, 20 to 50, 30 to 50 or 40 to 50, and a Thiol-pair bufferstrength (as defined herein) equal to or greater than 2 mM, for examplegreater than or equal to 2.25 mM, 2.5 mM, 2.75 mM, 3 mM, 5 mM, 7.5 mM,10 mM, or 15 mM, wherein the thiol-pair buffer strength is effectivelybounded at a maximum of 100 mM. Restated, in terms of ranges, the thiolbuffer strength is between 2 and 20 mM, for example between 2.25 mM and20 mM, 2.5 mM and 20 mM, 2.75 mM and 20 mM, 3 mM and 20 mM, 5 mM and 20mM, 7.5 mM and 20 mM, 10 mM and 20 mM, or 15 mM and 20 mM to form amixture. A wide range of denaturant types may be employed in the refoldbuffer. Examples of some common denaturants that can be employed in therefold buffer include urea, guanidinium, dimethyl urea, methylurea, orethylurea. The specific concentration of the denaturant can bedetermined by routine optimization, as described herein.

A wide range of protein stabilizers or aggregation suppressors can beemployed in the refold buffer. Examples of some common aggregationsuppressors that can be useful in the refold buffer include arginine,proline, polyethylene glycols, non-ionic surfactants, ionic surfactants,polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodiumsulfate, potassium sulfate, other osmolytes, or similar compounds. Thespecific concentration of the aggregation suppressor can be determinedby routine optimization, as described herein.

A redox component of the refold buffer can be of any composition, withthe caveat that the redox component has a final thiol-pair ratio in arange of 0.001 to 100, for example within a range of 0.05 to 50, 0.1 to50, 0.25 to 50, 0.5 to 50, 0.75 to 40, 1.0 to 50 or 1.5 to 50, 2 to 50,5 to 50, 10 to 50, 15 to 50, 20 to 50, 30 to 50 or 40 to 50, and aThiol-pair buffer strength of greater than or equal to 2 mM, for examplegreater than or equal to 2.25 mM, 2.5, 2.75 mM, 3 mM, 5 mM, 7.5 mM, 10mM, or 15 mM, wherein the thiol-pair buffer strength is effectivelybounded at a maximum of 100 mM. Restated, in terms of ranges, the thiolbuffer strength is between 2 and 20 mM, for example between 2.25 mM and20 mM, 2.5 mM and 20 mM, 2.75 mM and 20 mM, 3 mM and 20 mM, 5 mM and 20mM, 7.5 mM and 20 mM, 10 mM and 20 mM, or 15 mM and 20 mM. Methods ofidentifying a suitable redox component, i.e., determining appropriatethiol-pair ratios and redox buffer strengths, are known and/or areprovided herein. Examples of specific thiol pairs that can form theredox component can include one or more of reduced glutathione, oxidizedglutathione, cysteine, cystine, cysteamine, cystamine, andbeta-mercaptoethanol. Thus, a thiol-pair can comprise, for example,reduced glutathione and oxidized glutathione. Another example of a thiolpair is cysteine and cystamine. The redox component can be optimized asdescribed herein.

After the protein has been contacted with a redox component having therecited thiol pair ratio and redox buffer strength to form a refoldmixture, the refold mixture is then incubated for a desired period oftime. The incubation can be performed under non-aerobic conditions, asdefined herein. Non-aerobic conditions need not be completely free ofoxygen, only that no additional oxygen other than that present in theinitial system is purposefully introduced. The incubation period isvariable and is selected such that a stable refold mixture can beachieved with the desired analytical properties. An incubation periodcan be, for example, 1 hour, 4 hours, 12 hours, 24 hours, 48 hours, 72hours, or longer.

Due to the sensitivity of high concentration refolds to the level ofoxygen present in the system and the tendency for oxygen mass transferto be greater at small-scale, a methodology and/or apparatus can bedeveloped to control the oxygen levels and maintain non-aerobicconditions for the incubation step. In one embodiment, the procedure cancomprise the preparation, dispensing and mixing of all refold componentsunder a blanket of inert gas, such as nitrogen or argon, to avoidentraining levels of oxygen into the reaction. This approach isparticularly helpful in identifying an acceptable thiol-pair ratio. Inanother embodiment useful at scales of 15 liters or less, the headspaceof the refold reactor containing the protein and refold buffer can bepurged with an inert gas or a mixture of inert gas and air or oxygen,and the reaction vessel sealed and mixed at a low rotational speed forthe duration of the incubation time.

Following the incubation, the protein is isolated from the refoldmixture. The isolation can be achieved using any known proteinpurification method. If the protein comprises a Fc domain, for example,a Protein A column provides an appropriate method of separation of theprotein from the refold excipients. In other embodiments, various columnchromatography strategies can be employed and will depend on the natureof the protein being isolated. Examples include HIC, AEX, CEX and SECchromatography. Non-chromatographic separations can also be considered,such as precipitation with a salt, acid or with a polymer such as PEG(see, e.g., US 20080214795). Another alternative method for isolatingthe protein from the refold components can include dialysis ordiafiltration with a tangential-flow filtration system.

In another exemplary refolding operation, inclusion bodies obtained froma non-mammalian expression system are solubilized in the range of 10 to100 grams of protein per liter and more typically from 20-40 g/L forapproximately 10-300 min. The solubilized inclusion bodies are thendiluted to achieve reduction of the denaturants and reductants in thesolution to a level that allows the protein to refold. The dilutionresults in protein concentration in the range of 1 to 15 g/L in a refoldbuffer containing urea, glycerol or sucrose, arginine, and the redoxpair (e.g., cysteine and cystamine). In one embodiment the finalcomposition is 1-4 M urea, 5-40% glycerol or sucrose, 25-500 mMarginine, 0.1-10 mM cysteine and 0.1-10 mM cystamine. The solution isthen mixed during incubation over a time that can span from 1 hour to 4days.

As noted herein, the disclosed method is particularly useful forproteins expressed in bacterial expression systems, and moreparticularly in bacterial systems in which the protein is expressed inthe form of inclusion bodies within the bacterial cell. The protein canbe a complex protein. i.e., a protein that (a) is larger than 20,000 MW,or comprises greater than 250 amino acid residues, and (b) comprises twoor more disulfide bonds in its native form. When the protein isexpressed in an inclusion body it is likely that any disulfide bondfound in the protein's native form will be misformed or not formed atall. The disclosed method is applicable to these and other forms of aprotein of interest. Specific examples of proteins that can beconsidered for refolding using the disclosed methods include antibodies,which are traditionally very difficult to refold at high concentrationsusing typical refold methods due to their relatively large size andnumber of disulfide bonds. The method can also be employed to refoldother Fc-containing molecules such as peptibodies, and more generally torefold any fusion protein comprising an Fc domain fused to anotherprotein.

Another aspect of the disclosed method is its scalability, which allowsthe method to be practiced on any scale, from bench scale to industrialor commercial scale. Indeed, the disclosed method will find particularapplication at the commercial scale, where it can be employed toefficiently refold large quantities of protein.

The present disclosure will now be illustrated by reference to thefollowing examples, which set forth certain embodiments. However, itshould be noted that these embodiments are illustrative and are not tobe construed as restricting the invention in any way.

EXAMPLES

The Examples presented herein demonstrate that thiol-pair ratio andredox buffer strength is a significant consideration in achieving anefficient refolding reaction that is insensitive to environmentalinfluences and aeration. This insensitivity is a consideration for theease of scaling and on an industrial or commercial scale, the transferof the process from plant to plant.

The Examples also demonstrate that at typical refolding reactionconcentrations (0.01-2.0 g/L); the sensitivity to external aeration isrelatively muted. However, at concentrations of about 2 g/L and above,the sensitivity of the refold reaction to the thiol pair ratio and redoxbuffer strength is increased and nearly all of the chemical components,especially the redox components, may need to be adjusted to accommodatefor changes in the protein concentration in the reaction.

Example 1 Expression of Recombinant Protein

In one experiment, recombinant proteins comprising an Fc moiety wereexpressed in a non-mammalian expression system, namely E coli, anddriven to form cytoplasmic deposits in the form of inclusion bodies. Foreach protein refolded the following procedure was followed.

After the completion of the expression phase, the cell broth wascentrifuged and the liquid fraction removed, leaving the cells as apaste. The cells were resuspended in water to approximately 60% of theoriginal volume. The cells were then lysed by means of three passesthrough a high pressure homogenizer. After the cells were lysed, thelysate was centrifuged in a disc-stack centrifuge to collect the proteinin the solid fraction, which was expressed in a limited solubilitynon-native form, namely as inclusion bodies. The protein slurry waswashed multiple times by repeatedly resuspending the captured solidsslurry in water to between 50% and 80% of the original fermentationbroth volume, mixing, and centrifugation to collect the protein in thesolid fraction. The final washed inclusion bodies were captured andstored frozen.

Example 2 Identification of Refold Conditions/Redox Components

Multiple complex, microbial-derived proteins were evaluated. Eachprotein was solubilized in an appropriate level of guanidine and/orurea, typically at levels the equivalent of 4-6 M guanidine or 4-9 Murea, or combinations of both denaturants, which fully denatured theprotein. The protein was reduced with DTT, 5-20 mM, at pH 8.5, andincubated at room temperature for approximately 1 hour.

Identification of the refold buffer was performed for each protein. Amultifactorial matrix or a series of multifactorial matrices wereevaluated to identify the refolding reaction for conditions thatoptimize yield and minimize aggregate formation. An identificationscreen was set up to systematically evaluate urea, arginine, glyceroland pH in a full factorial matrix, with each component varied over arange of at least three concentration or pH levels with all otherparameters kept constant. The completed reactions were evaluated byRP-HPLC and SE-HPLC analysis for yield and product quality usingstandard multivariate statistical tools. A subset of the conditionshaving the desired behavior was then further evaluated in subsequentscreens that evaluated a range of pH, thiol-pair ratio, thiol-pairbuffer strength, and potentially further excipient levels in a factorialscreen. Secondary interactions were also evaluated using standardmultivariate statistical tools.

Best results, as determined by reversed-phase and size exclusion HPLCanalysis, were observed using a refold buffer containing a denaturant(e.g., urea, dimethyl urea or other chaotrope at non-denaturing levelsat levels between 1 and 4 M), an aggregation suppressor (e.g., arginineat levels between 5 and 500 mM), a protein stabilizer (e.g., glycerol orsucrose at levels between 5 and 40% w/v) and a redox component (e.g.,cysteine or cystamine). The thiol-pair ratio and redox buffer strengthwere determined using an experimental matrix of thiol-pair ratio (0.1 to100, more typically 1 to 25) versus buffer strength (typically 2 mM to20 mM, depending on the protein concentration, the number of cysteineresidues in the protein, and the concentration of reductant used tosolubilize the inclusion bodies).

Individual reactions were formed with varying levels of cysteine andcystamine that would allow for a controlled matrix of thiol-pair ratioat various thiol-pair buffer strengths. The relationships werecalculated using Equations 3 and 4. Each condition was screened underboth aerobic and non-aerobic conditions, utilizing the techniquesdescribed herein. Optimum conditions were selected to meet a stablebalance of yield, desired distribution of folding species, insensitivityto environmental oxidants (e.g., air), and insensitivity to normalvariation in DTT carry-over from the solubilization step.

Example 3 High Concentration Refolding of Non-Native Soluble ProteinForm Captured from Cell Lysate

In one experiment, a recombinant protein comprising a plurality ofpolypeptides joined to an Fc moiety was expressed in E. coli as anintracellular soluble peptide chain, lysed from harvested and washedcells, isolated from the lysate by affinity chromatography, and thenrefolded at a concentration of approximately 12 g/L, as describedherein.

After the completion of the expression phase, an aliquot of wholefermentation broth was centrifuged and the liquid fraction removed,leaving the cells as a paste. The cells were resuspended in water toapproximately 60% of the original volume. The cells were then lysed bymeans of three passes through a high pressure homogenizer. After thecells were lysed, the lysate pool was mixed in the presence of air for8-72 hours to allow for dimerization of the peptide chains. Followingthe dimerization process, the peptide chain of interest was isolatedfrom the lysate pool using a Protein A affinity chromatography column.The Protein A column elution pool was mixed at a ratio of 8 partsProtein A elution material to 2 parts of a refold buffer containing urea(10 M), arginine-HCl (2.5 M), Tris at pH 8.5 (1050 mM), and cysteine (10mM, 5 mM, or 4 mM) and cystamine (4 mM). The diluted mixture wastitrated to pH 8.5 and incubated at approximately 5° C. under nitrogenuntil a stable pool was achieved (˜24 hours.) Yields of desired productof approximately 30-80% were obtained a depending on the redox conditionevaluated.

In order to emulate the non-anaerobic conditions similar to thosetypically present in very large-scale protein production processesseveral steps were taken. When reaction volumes were less thanapproximately 15 L the refold vessel headspace was purged with nitrogento limit the effect oxygen could have in the system. The vessel was thensealed and incubation began.

When reaction volumes were more than approximately 15 L but less than500 L, the refold buffer was prepared and allowed to equilibrate atapproximately 5° C. to achieve a stable oxygen level in the solution(typically 50% to 70% dissolved oxygen, relative to air saturation).Once the refold mixture was formed, the vessel headspace was purged withnitrogen to limit any additional effect oxygen could have in the system,the vessel was sealed and incubation period initiated.

Example 4 Concentration Refolding from Inclusion Bodies

In one experiment, a recombinant protein comprising a biologicallyactive peptide linked to the C-terminus of the Fc moiety of an IgG1molecule via a linker and having a molecular weight of about 57 kDa andcomprising 8 disulfide bonds, was expressed in E. coli as inclusionbodies, harvested, washed, concentrated, solubilized, and refolded at aconcentration of 6 g/L as described herein.

An aliquot of frozen concentrated inclusion bodies were thawed to roomtemperature and mixed with an appropriate amount of guanidine and/orurea to generate a denaturant level equivalent to 4-6 M guanidine, whichfully denatures the protein. The protein was then reduced with DTT, at5-20 mM, at pH 8.5, and incubated at room temperature for approximately1 hour. After the inclusion bodies were dissolved, denatured andreduced, they were diluted into a refold buffer containing urea (1-5 M),arginine-HCl (5-500 mM), glycerol (10-30% w/v), and the identifiedlevels of cysteine and cystamine as determined by the proceduredescribed in Example 2. The final component concentrations are 4 M urea,150 mM arginine HCl, 20.9% (w/v) glycerol, 2.03 mM cysteine, and 2.75 mMcystamine. The level of dilution was chosen to balance the dilution ofthe denaturants from the solubilization, maintain the thermodynamicstability of the molecule during refolding, and maintain the highestpossible protein concentration in the refold mixture. The dilutedmixture was titrated to an alkaline pH (between pH 8 and pH 10) andincubated at 5° C. under non-aerobic conditions until a stable pool wasachieved (12-72 hours), as determined by relevant analyticalmeasurements. The resulting process was demonstrated to show stablescalablity from 1 L-scale to 2000 L-scale (see FIG. 3). Yields ofdesired product of approximately 27-35% were obtained at both scales.The distribution of product related impurities was also maintainedwithin a tight variance (see FIG. 3).

Oxygen mass transfer at small-scale is readily achieved and should beinhibited in order to emulate the relatively poorer mass transferobserved at large-scale, where the volume of refold solution is largerelative to the volume of air and surface area present at the surface ofa large-scale vessel. Thus, in order to emulate the non-anaerobicconditions similar to those typically present in very large-scaleprotein production processes several steps were taken. When reactionvolumes were less than approximately 15 L the refold buffer was spargedwith nitrogen to strip oxygen from the solution, the components weredispensed under a blanket of nitrogen and once the refold mixture wasformed, the vessel headspace was purged with nitrogen to limit theeffect oxygen could have in the system. The vessel was then sealed andincubation began.

When reaction volumes were more than approximately 15 L but less than500 L, the refold buffer was prepared and allowed to equilibrate atapproximately 5° C. to achieve a stable oxygen level in the solution(typically 50% to 70% dissolved oxygen, relative to air saturation).Once the refold mixture was formed, the vessel headspace was purged withnitrogen to limit any addition effect oxygen could have in the system,the vessel was sealed and the incubation period was initiated.

At scales greater than 500 L the refold buffer was prepared and allowedto equilibrate at approximately 5° C. to achieve a stable oxygen levelin the solution (typically 50% to 70% dissolved oxygen, relative to airsaturation). Once the refold mixture was formed, the vessel was sealedand the incubation period was initiated.

The protein concentration of the refold mixture was 6 g/L, which is afour-fold enhancement over the recovery of 1.5 g/L obtained using amethod other than the method described in this Example. Overall annualprocess productivity, in one specific manufacturing facility, wascalculated to be increased by >930% due to increased volumetricefficiency in the existing facility tanks.

Example 5 Effect of Thiol-Pair Oxidation State on Disulfide Pairings

FIGS. 1 a-1 f demonstrate that as the thiol-pair ratio is forced to amore oxidizing state (lower thiol-pair ratio), a higher proportion ofproduct species have oxidized amino acid residues and mixed disulfideforms. As the thiol-pair ratio is driven to a more reductive state(higher thiol-pair ratio), this results in lower levels of oxidizedamino acid variant species and higher levels of product species withincorrect disulfide pairings or unformed disulfide bonds. As the overallthiol-pair buffer strength is modified, the corresponding optimalthiol-pair ratio is shifted. This effect is similar to how bufferstrength modulates the sensitivity of pH to acid and base additions in abuffered solution.

An optimal balance of species was attainable. As shown in FIGS. 1 a-1 f,there is a clear relationship between thiol-pair buffer strength andthiol-pair ratio that can be identified to maintain the optimal speciesbalance and thus facilitate efficient refolding of low solubilityproteins. The ability to control product variant species, such asincorrectly disulfide-bonded species and misfolded species, viamodulation of the thiol-pair ratio and thiol-pair buffer strength,enables efficient, effective and reliable subsequent purificationprocesses.

Example 6 Effect of Non-Aerobic Conditions on Refolding Efficiency

FIGS. 2 and 3 demonstrate that when the thiol-pair buffer strength isselected appropriately, taking into account the protein concentrationand number of cysteine residues in the protein, the sensitivity toexternal influences, such as oxygen, is significantly reduced. Thisallows for a non-aerobic refolding condition that is significantlyeasier to transfer between scales and reactor configurations.

FIG. 2 compares the RP-HPLC analytical species distribution between a 15L-scale refold and a 20 mL-scale refold under several environmentalconditions. For Condition 1 (the trace labeled “1” in FIG. 1), thesolubilization chemicals and solutions were dispensed in air and therefold mixture was incubated in air. In Condition 2 solubilizationchemicals and solutions were dispensed in air and incubated undernitrogen headspace. In Conditions 3-7 solubilization chemicals andsolutions were dispensed under nitrogen overlay conditions and inconditions 3, 5, 6, and 7 solubilization chemicals and solutions wereincubated under nitrogen. In Condition 7, the refold solution was alsostripped of nitrogen prior to combination with the sobibilizationsolution. In Condition 4 the solubilization chemicals and solutions wereincubated under ambient air conditions.

The results shown in FIG. 2 demonstrate that the conditions under whichthe solubilization chemicals and solutions were dispensed or incubatedin the presence of air (i.e., Conditions 1, 2, and 4) do not achieveresults that are comparable to the larger-scale control. In Conditions1, 2 and 4, increased formation of oxidized species (pre-peaks) areobserved. The pre-peaks are indicated by arrows in the panels forConditions 1, 2 and 4.

FIG. 3 compares the RP-HPLC analytical results of an identifiedcondition, achieved as described in Example 2, at 1 L-scale and 2000L-scale. In this figure, essentially no difference in the distributionof species is detectable. Taken together, FIGS. 2 and 3 demonstrate thatwhen aeration is carefully controlled, the small-scale refold reactionsare more predictive of those expected upon scale-up of the refoldreaction, facilitating the implementation of large-scale proteinrefolding processes.

1-24. (canceled)
 25. A method of refolding a protein expressed in anon-mammalian expression system, said method comprising: (a) contactingsaid protein with a refold buffer to form a refold mixture, wherein saidprotein is present at a concentration of 2.0 g/L or greater in saidrefold mixture, and wherein said refold buffer comprises: (i) a redoxcomponent comprising a final thiol-pair ratio, wherein said finalthiol-pair ratio has a range of 0.001 to 50, and wherein said thiol-pairratio is calculated according to Equation 1:$\frac{\lbrack{reductant}\rbrack^{2}}{\lbrack{oxidant}\rbrack};$ (ii) athiol pair buffer strength of 2 mM or greater, wherein said thiol-pairbuffer strength is calculated according to Equation 2:2[oxidant]+[reductant]; and (iii) at least one of: a denaturant; anaggregation suppressor; and a protein stabilizer; and (b) incubatingsaid refold mixture.
 26. The method of claim 25, wherein said methodfurther comprises: (c) isolating the refolded protein from step b). 27.The method of claim 25, wherein said protein is present at aconcentration of 2 to 40 g/L in said refold mixture.
 28. The method ofclaim 27, wherein said protein is present at a concentration of 10 to 20g/L in said refold mixture.
 29. The method of claim 25, wherein saidrefold buffer comprises a denaturant, an aggregation suppressor, and aprotein stabilizer.
 30. The method of claim 25, wherein said protein isan antibody.
 31. The method of claim 25, wherein said protein comprisesan Fc domain.
 32. The method of claim 25, wherein said final thiol-pairratio has a range of 1 to
 25. 33. The method of claim 25, wherein saidthiol-pair buffer strength is greater than or equal to 5 mM.
 34. Themethod of claim 25, wherein said protein in step a) is present in anon-native limited solubility form at the time said protein is contactedwith said refold buffer.
 35. The method of claim 34, wherein saidnon-native limited solubility form is an inclusion body.
 36. The methodof claim 25, wherein said incubation is performed under non-aerobicconditions.
 37. The method of claim 25, wherein said non-mammalianexpression system is a bacterial expression system or a yeast expressionsystem.
 38. The method of claim 37, wherein said non-mammalianexpression system is a bacterial expression system.
 39. The method ofclaim 38, wherein said bacterial expression system is an E. coliexpression system.
 40. A method of refolding a protein expressed in anE. coli expression system, said method comprising: (a) contacting saidprotein with a refold buffer to form a refold mixture, wherein saidprotein is present at a concentration of 2 to 40 g/L in said refoldmixture, and wherein said refold buffer comprises: (i) a redox componentcomprising a final thiol-pair ratio, wherein said final thiol-pair ratiohas a range of 1 to 50, and wherein said thiol-pair ratio is calculatedaccording to Equation 1:$\frac{\lbrack{reductant}\rbrack^{2}}{\lbrack{oxidant}\rbrack};$ (ii) athiol pair buffer strength between 2 mM and 20 mM, wherein saidthiol-pair buffer strength is calculated according to Equation 2:2[oxidant]+[reductant]; and (iii) at least one of: a denaturant; anaggregation suppressor; and a protein stabilizer; and (b) incubatingsaid refold mixture incubation under non-aerobic conditions.
 41. Themethod of claim 40, wherein said method further comprises: (c) isolatingthe refolded protein from step b).
 42. The method of claim 40, whereinsaid refold buffer comprises a denaturant, an aggregation suppressor,and a protein stabilizer.
 43. The method of claim 40, wherein saidprotein is present at a concentration of 10 to 20 g/L in said refoldmixture.
 44. The method of claim 40, wherein said protein is anantibody.
 45. The method of claim 40, wherein said protein comprises anFc domain.
 46. The method of claim 40, wherein said final thiol-pairratio has a range of 1 to
 25. 47. The method of claim 40, wherein saidthiol-pair buffer strength is between 5 mM and 20 mM.
 48. The method ofclaim 47, wherein said thiol-pair buffer strength is between 10 mM and20 mM.
 49. The method of claim 40, wherein said protein in step a) ispresent in a non-native limited solubility form at the time said proteinis contacted with said refold buffer.
 50. The method of claim 49,wherein said non-native limited solubility form is an inclusion body.51. The method of claim 40, wherein said denaturant is selected from thegroup consisting of urea, guanidinium salts, dimethyl urea, methylureaand ethylurea.
 52. The method of claim 40, wherein said aggregationsuppressor is selected from the group consisting of arginine, proline,polyethylene glycols, non-ionic surfactants, ionic surfactants,polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodiumsulfate, potassium sulfate and osmolytes.
 53. The method of claim 40,wherein said protein stabilizer is selected from the group consisting ofarginine, proline, polyethylene glycols, non-ionic surfactants, ionicsurfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose,Tris, sodium sulfate, potassium sulfate and osmolytes.
 54. The method ofclaim 40, wherein said thiol-pairs comprise at least one thiol-pairselected from the group consisting of reduced glutathione, oxidizedglutathione, cysteine, cystine, cysteamine, cystamine andbeta-mercaptoethanol.